Strains and proteins derived from Bacillus thuringiensis (abbreviated herein as “Bt”) are well known for their specific toxicity to insect pests, and they have been used since almost a century to control insect pests. Some transgenic plant species expressing Bt proteins are now available, and they successfully limit insect damage on plants.
Despite the isolation of quite a number of insecticidal Bt proteins, only a few Bt proteins have been expressed in transgenic plants that have been commercialized, and this only in some crops.
Insect pests are important constraints for rice production and occur in all rice growing environments. Insects reduce rice yields substantially and losses due to insects in Asia (excluding China) have been reported to be about 31.5% (Heinrichs, 1994). Insect resistance in rice has been reported by expressing genes encoding insecticidal Cry1Ab or Cry1Ac proteins of Bacillus thuringiensis (e.g., Fujimoto et al., 1993; Wunn et al., 1996; Wu et al. 1997; Ghareyazie et al., 1997; Nayak et al., 1997; and Cheng et al., 1998) in rice plants. Toxicity of isolated Bacillus thuringiensis crystal proteins to some Lepidopteran rice insect pests has been evaluated for some proteins of the classes of Bt proteins Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, and Cry2A (Karim et al., 1997; Lee et al., 1997), but several different forms of each of these protein classes exist (e.g., today about 14 different Cry1C and about 20 different Cry1Ab forms have been reported in Crickmore et al. (1998) and in Crickmore et al. (2005)), and only one or a few of these were tested against rice insect pests. It is believed that no scientific publication describes the activity of the Cry1Ca4 protein to specific rice insect pests.
Also, Strizhov et al. (1998) describe the design of a synthetic gene encoding a Cry1Ca5 protein. After analyzing and comparing with the known proteins (Cry1Ca1, 2, 3, and 4; on page 18, line 3 to page 19, line 6) they conclude that the sequence of the Cry1Ca5 protein differs by amino acid replacement A124E from the Cry1Ca4 protein. Strizhov et al. (1998) conclude that the occurrence of glutamic acid at position 124 in Cry1Ca4 was clearly due to an error. Strizhov et al. (1998) also believe that the then known Cry1C sequences, including Cry1Ca4, contain critical errors with negative consequences either for function or stability of the Cry1C protein, had a corresponding synthetic gene been designed on the basis of the known wild-type DNA sequences. Yet, the current inventors succeeded in producing useful synthetic cry1C genes encoding an insecticidal Cry1C protein having a glutamic acid (Glu) amino acid at position 124, and in obtaining plants resistant to insects using such genes.
No rice plants containing a cry1C gene are commercially available. Tang et al. (2006) describe the development of insect-resistant transgenic indica rice with a synthetic gene encoding a Cry1Ca5 protein, but do not disclose the DNA sequence of the gene. The gene used in Tang et al. (2006) is said to be 84% identical to the native cry1Ca5 DNA, while the cry1C coding sequence of this invention is 69.7% identical to the native cry1Ca5 DNA of the same length when measured using the Needleman and Wunsch algorithm in EMBOSS with standard settings for the EDNAFULL matrix, and hence is quite different from the cry1Ca5 gene of Tang et al. (2006) (for the cry1C DNA sequence of SEQ ID No. 1, the sequence identity with the same length native cry1Ca5 DNA is only 55%, under the same settings). Also, the PCR primer set provided in Tang et al. (2006) will not allow detection of the cry1C coding region of the invention, illustrating the clear difference between that gene and the cry1C genes of the invention.
Strizhov et al. (1996) report the expression of a cry1Ca5 gene in alfalfa and tobacco. Cao et al. (1999) and Zhao et al. (2003) have described transgenic broccoli plants expressing a Cry1Ca5 Bt toxin, as well as crosses with broccoli plants expressing a Cry1Ac toxin, so that both the Cry1Ac and Cry1Ca5 toxins are expressed in the same plants. The cry1C coding sequence of this invention is only 78% identical to the coding sequence of the synthetic cry1Ca5 gene of Strizhov et al. (1996, Genbank accession number X99103) when measured using the Needleman and Wunsch algorithm in EMBOSS with standard settings for the EDNAFULL matrix (the DNA of SEQ ID No. 1 only has 61.1% sequence identity to the synthetic cry1Ca5 gene of Strizhov et al. using the same standard settings in EMBOSS).
The current invention provides new synthetic genes encoding a protein derived from the Cry1Ca4 Bt protein, which can be combined with a gene encoding a Bt Cry protein, such as Cry1Ab protein, for expression in plants, particularly rice. The DNA sequence of the cry1C genes of the invention do not occur in nature, and are different from any known DNA sequence.